Reagent test strip for analyte determination having a hemolyzing agent

ABSTRACT

Reagent test strips and methods for their use in the determination of the concentration of an analyte, e.g. glucose, in a physiological sample, are provided. The subject reagent test strips include one or more members of an analyte oxidation signal producing system and at least one hemolyzing agent. The subject reagent test strips and methods are particularly suited for use in the detection of blood glucose concentrations. Also provided are kits that include the subject test strips for use in practicing the subject methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/155,949, filed on May 24, 2002, now allowed which is a divisional ofU.S. patent application Ser. No. 09/497,631, filed on Feb. 2, 2000, nowissued U.S. Pat. No. 6,485,923.

FIELD OF THE INVENTION

The field of this invention is analyte determination, particular bloodanalyte determination and more particularly blood glucose determination.

BACKGROUND

Analyte detection in physiological fluids, e.g. blood or blood derivedproducts such as plasma, is of ever increasing importance to today'ssociety. Analyte detection assays find use in a variety of applicationsand settings, including the clinical laboratory testing, home testing,etc., where the results of such testing play a prominent role indiagnosis and management in a variety of disease conditions. Analytes ofinterest include glucose for diabetes management, cholesterol formonitoring cardiovascular conditions, and the like. In response to thisgrowing importance of analyte detection, a variety of analyte detectionprotocols and devices for both clinical and home use have beendeveloped.

Many analyte detection assays are based on the production of hydrogenperoxide and the subsequent detection thereof. Analytes that may bedetected using such assays include: cholesterol, triglycerides, glucose,ethanol and lactic acid. For example, glucose is quantitated using suchassays by first oxidizing glucose with glucose oxidase to producegluconic acid and hydrogen peroxide. The resultant hydrogen peroxide, inconjunction with a peroxidase, causes the conversion of one or moreorganic substrates, i.e. an indicator, into a chromogenic product, whichproduct is then detected and related to the glucose concentration in theinitial sample.

Hydrogen peroxide based assays, such as the glucose assay describedabove, are subject to problems which result from the presence oferythrocyte components, e.g. catalase, that interfere with the hydrogenperoxide based reaction and therefore alter (for example reduce) thesignal that is ultimately obtained and used to derive the analyteconcentration. As such, many different protocols have been developedwhich are designed to at least reduce the potential analytical errorthat is introduced in the assay through the release of interferingerythrocyte components via hemolysis. Such protocols include:filtration, filtration combined with the addition of inhibitors,filtration and trapping of erythrocytes, and the use of asymmetricnon-hemolyzing membranes.

While such methods can partially remove the analytical error introducedby hemolysis, they are not entirely satisfactory. For example,filtration typically requires longer assay times and larger sample sizesthan is desirable.

As such, there is continued interest in the development of new devicesand methods for use in analyte detection. Of particular interest wouldbe the development such a device and method which minimized theanalytical error originating from hemolysis and yet provided a rapidassay time from a small sample volume.

Relevant Literature

U.S. patent documents of interest include: U.S. Pat. Nos. 4,297,238;5,258,047; 5,563,042; 5,753,452; 5,789,255; 5,843,691; 5,866,349;5,968,836 and 5,972,294. Also of interest are: WO 90/12889; WO 90/12890;JP 3180762; JP 62296987; and EP 0 638 805.

SUMMARY OF THE INVENTION

Reagent test strips and methods for their use in the determination ofthe concentration of an analyte, e.g. glucose, in a physiological sampleare provided. The subject reagent test strips include one or moremembers of an analyte oxidation signal producing system and at least onehemolyzing agent. The subject reagent test strips and methods areparticularly suited for use in the detection of blood glucoseconcentrations. Also provided are kits that include the subject teststrips for use in practicing the subject methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a graphical representation of the effect of hemolysateon test response.

FIG. 2 a provides a graphical representation of the effect of hematocriton test response in the absence of a hemolyzing agent, while FIG. 2 bprovides a graphical representation of the effect of hematocrit on testresponse in the presence of 0.25% CTAC.

FIGS. 3 and 4 provide graphical representations of the test response andreaction kinetics observed at a whole blood glucose concentration of 390mg/dL in the absence and presence of 0.25% CTAC.

FIGS. 5 and 6 provide graphical representations of the test response andreaction kinetics observed at a whole blood glucose concentration of 390mg/dL in the absence and presence of 0.25% Triton X-100.

FIGS. 7 and 8 provide graphical representations of the test response andreaction kinetics observed at a whole blood glucose concentration of 390mg/dL in the absence and presence of 0.50% Brij-58.

FIG. 9 provides a graphical representation of the test response andreaction kinetics observed at a whole blood glucose concentration of 390mg/dL in the presence of 0.50% Lubrol PX.

FIG. 10 provides a graphical representation of the variation in observedK/S in the presence and absence of 0.25% CTAC in 60% Hct blood having a0.0 mg/dL glucose concentration.

FIG. 11 provides a graphical representation of the variation in observedK/S in the presence and absence of 0.25% CTAC in 60% Hct blood having a30 mg/dL glucose concentration.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Reagent test strips for use in the determination of the concentration ofan analyte, e.g. glucose, in a physiological sample, e.g. blood, areprovided. The subject test strips include a porous matrix, one or moremembers of an analyte oxidation signal producing system and at least onehemolyzing agent. In using the subject test strips for analyteconcentration determination, a physiological sample is applied to thetest strip. Next, the appearance of a chromogenic product of the signalproducing system is detected and related to the concentration of theanalyte in the sample. Also provided by the subject invention are kitsfor practicing the subject methods, where the kits at least include thesubject reagent test strips. In further describing the subjectinvention, the subject test strips and methods for their use arediscussed in greater detail, followed by a review of the subject kits.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, singular referencesinclude the plural, unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

REAGENT TEST STRIPS

As summarized above, the reagent test strips of the subject inventionare characterized by having at least the following components: a porousmatrix; one or more members of an analyte oxidation signal producingsystem; and at least one hemolyzing agent. Each one of these componentsis now described separately in greater detail.

The Porous Matrix

The matrix that is employed in the subject test strips is an inertporous matrix which provides a support for the various members of thesignal producing system, described infra, as well as the light absorbingor chromogenic product produced by the signal producing system, i.e. theindicator. The inert porous matrix is configured to provide a locationfor physiological sample, e.g. blood, application and a location fordetection of the light-absorbing product produced by the indicator ofthe signal producing system. As such, the inert porous matrix is onethat is permissive of aqueous fluid flow through it and providessufficient void space for the chemical reactions of the signal producingsystem to take place. A number of different porous matrices have beendeveloped for use in various analyte detection assays, which matricesmay differ in terms of materials, pore sizes, dimensions and the like,where representative matrices include those described in: U.S. Pat. Nos.4,734,360; 4,900,666; 4,935,346; 5,059,394; 5,304,468; 5,306,623;5,418,142; 5,426,032; 5,515,170; 5,526,120; 5,563,042; 5,620,863;5,753,429; 5,573,452; 5,780,304; 5,789,255; 5,843,691; 5,846,486;5,968,836 and 5,972,294; the disclosures of which are hereinincorporated by reference. In principle, the nature of the porous matrixis not critical to the subject test strips and therefore is chosen withrespect to the other factors, including the nature of the instrumentwhich is used to read the test strip, convenience and the like. As such,the dimensions and porosity of the test strip may vary greatly, wherethe matrix may or may not have a porosity gradient, e.g. with largerpores near or at the sample application region and smaller pores at thedetection region. Materials from which the matrix may be fabricatedvary, and include polymers, e.g. polysulfone, polyamides, cellulose orabsorbent paper, and the like, where the material may or may not befunctionalized to provide for covalent or non-covalent attachment of thevarious members of the signal producing system, described in greaterdetail infra.

The Signal Producing System

In addition to the porous matrix, the subject test strips furtherinclude one or more members of a signal producing system which producesa detectable product in response to the presence of analyte, whichdetectable product can be used to derive the amount of analyte presentin the assayed sample. In the subject test strips, the one or moremembers of the signal producing system are associated, e.g. covalentlyor non-covalently attached to, at least a portion of (i.e. the detectionregion) the porous matrix, and in many embodiments to substantially allof the porous matrix.

The signal producing system is an analyte oxidation signal producingsystem. By analyte oxidation signal producing system is meant that ingenerating the detectable signal from which the analyte concentration inthe sample is derived, the analyte is oxidized by a suitable enzyme toproduce an oxidized form of the analyte and a corresponding orproportional amount of hydrogen peroxide. The hydrogen peroxide is thenemployed, in turn, to generate the detectable product from one or moreindicator compounds, where the amount of detectable product producing bythe signal producing system, i.e. the signal, is then related to theamount of analyte in the initial sample. As such, the analyte oxidationsignal producing systems present in the subject test strips are alsocorrectly characterized as hydrogen peroxide based signal producingsystems.

As indicated above, the hydrogen peroxide based signal producing systemsinclude an enzyme that oxidizes the analyte and produces a correspondingamount of hydrogen peroxide, where by corresponding amount is meant thatthe amount of hydrogen peroxide that is produced is proportional to theamount of analyte present in the sample. The specific nature of thisfirst enzyme necessarily depends on the nature of the analyte beingassayed but is generally an oxidase. As such, the first enzyme may be:glucose oxidase (where the analyte is glucose); cholesterol oxidase(where the analyte is cholesterol); alcohol oxidase (where the analyteis alcohol); lactate oxidase (where the analyte is lactate) and thelike. Other oxidizing enzymes for use with these and other analytes ofinterest are known to those of skill in the art and may also beemployed. In those preferred embodiments where the reagent test strip isdesigned for the detection of glucose concentration, the first enzyme isglucose oxidase. The glucose oxidase may be obtained from any convenientsource, e.g. a naturally occurring source such as Aspergillus niger orPenicillum, or recombinantly produced.

The second enzyme of the signal producing system is an enzyme thatcatalyzes the conversion of one or more indicator compounds into adetectable product in the presence of hydrogen peroxide, where theamount of detectable product that is produced by this reaction isproportional to the amount of hydrogen peroxide that is present. Thissecond enzyme is generally a peroxidase, where suitable peroxidasesinclude: horseradish peroxidase (HRP), soy peroxidase, recombinantlyproduced peroxidase and synthetic analogs having peroxidative activityand the like. See e.g., Y. Ci, F. Wang; Analytica Chimica Acta, 233(1990), 299–302.

The indicator compound or compounds, e.g. substrates, are ones that areeither formed or decomposed by the hydrogen peroxide in the presence ofthe peroxidase to produce an indicator dye that absorbs light in apredetermined wavelength range. Preferably the indicator dye absorbsstrongly at a wavelength different from that at which the sample or thetesting reagent absorbs strongly. The oxidized form of the indicator maybe the colored, faintly-colored, or colorless final product thatevidences a change in color of the testing side of the membrane. That isto say, the testing reagent can indicate the presence of glucose in asample by a colored area being bleached or, alternatively, by acolorless area developing color.

Indicator compounds that are useful in the present invention includeboth one- and two-component chromogenic substrates. One-componentsystems include aromatic amines, aromatic alcohols, azines, andbenzidines, such as tetramethyl benzidine-HCl. Suitable two-componentsystems include those in which one component is MBTH, an MBTH derivative(see for example those disclosed in U.S. patent application Ser. No.08/302,575, incorporated herein by reference), or 4-aminoantipyrine andthe other component is an aromatic amine, aromatic alcohol, conjugatedamine, conjugated alcohol or aromatic or aliphatic aldehyde. Exemplarytwo-component systems are 3-methyl-2-benzothiazolinone hydrazonehydrochloride (MBTH) combined with 3-dimethylaminobenzoic acid (DMAB);MBTH combined with 3,5-dichloro-2-hydroxybenzene-sulfonic acid (DCHBS);and 3-methyl-2-benzothiazolinone hydrazone N-sulfonyl benzenesulfonatemonosodium (MBTHSB) combined with 8-anilino-1 naphthalene sulfonic acidammonium (ANS). In certain embodiments, the dye couple MBTHSB-ANS ispreferred.

In yet other embodiments, signal producing systems that produce afluorescent detectable product (or detectable non-fluorescent substance,e.g. in a fluorescent background) may be employed, such as thosedescribed in:

Kiyoshi Zaitsu, Yosuke Ohkura: New fluorogenic substrates forHorseradish Peroxidase: rapid and sensitive assay for hydrogen peroxideand the Peroxidase. Analytical Biochemistry (1980) 109, 109–113.

Hemolyzing Agent

A feature of the subject reagent test strips is the presence of at leastone hemolyzing reagent. By hemolyzing agent is meant an agent that iscapable of lysing erythrocytes or red blood cells. Any convenienthemolyzing agent may be employed, where a variety of differenthemolyzing agents are known to those of skill in the art. Representativehemolyzing agents of interest include ionic surface-active agents, bothanionic and cationic, and non-ionic surface active agents, whereparticular surfactants of interest include: sodium dodecylsulfate,cetyltrimethylammonium bromide, laurylsarcosine or tauroglycocholate,alkylphenol polyglycol ethers, e.g. polyoxyethylene-10-octylphenol ether(Triton® X 100), polyoxyethylene-7.8-octylphenol ether (Triton® X 114),polyoxyethylene-10-nonylphenol ether (Renex®690),polyoxyethylene-9-nonylphenol ether (Renex® 680); N-hexadecyltrimetheylammonium chloride; [Brij-58; Lubrol PX] polyoxyethylene 20 cetyl ether(Brij® 58); polyoxyethylene 9 lauryl ether (Lubrol® PX), and the like.Other agents of interest include: phospholipases, hemolyzing saponins,compounds of hydrophilic mono-, di-, or trisaccharides and aliphatichydrocarbons having 10 to 16 carbon atoms (See e.g. PCT/5E90/00272, thedisclosure of which is herein incorporated by reference) colloidalsilica, silicic acid, hydroxyapatite crystals, and the like.

The subject test strips may include one type of hemolyzing agent, or mayinclude two or more different types of hemolyzing agents, e.g. aplurality of different hemolyzing agents. Where the subject test stripsinclude more than one hemolyzing agent, i.e. a plurality of hemolyzingagents, the strips generally include from two to five differenthemolyzing agents, and usually from two to four different hemolyzingagents. The total amount of the one or more hemolyzing agents that isincluded in the test strip is chosen to produce hemolysis which isequivalent to at least about 5% hemolysate by volume in the sampleusually at least about 8% and in many embodiments at least about 10%hemolysate in the sample, e.g. plasma fraction, that is ultimatelypresent in the detection region following sample application. In certainembodiments, the amount of hemolyzing agent(s) present in the test stripis sufficient to provide from about 5 to 40, usually from about 8 to 30and more usually from about 10 to 20% (v/v) hemolysate in the sample,e.g. plasma fraction, that is present in the detection region of thestrip during use. The amount of hemolyzing agent required to yield therequisite hemolysate in the sample may readily be determined empiricallyby those of skill in the art.

The reagent test strips of the subject invention can be prepared usingany convenient method. One convenient means of preparing the subjecttest strips is to immerse a porous matrix into to one or more fluidcompositions that comprise the various reagents that are to beassociated with the matrix in the final test strip. The fluidcompositions are generally aqueous compositions that include one or moreof the requisite reagents and, optionally, other components, includingcosolvents (e.g. organic cosolvents such as methanol, ethanol isopropylalcohol) and the like. In such embodiments, the concentration ofoxidase, e.g. glucose oxidase, in the fluid composition into which theporous matrix is immersed or dipped typically ranges from about 1500U/mL to 800 U/mL, usually from about 990 U/mL to 970 U/mL; theconcentration of peroxidase typically ranges from about 1500 U/mL to 800U/mL and usually from about 1050 U/mL to 900 U/mL; and the concentrationof hemolyzing agent(s) typically ranges from about 0.1% (w/v) to 0.5%(w/v), usually from about 0.15% (w/v) to 0.25% (w/v). A more detailedrepresentative protocol on how to prepare the subject reagent teststrips is provided in the Experimental Section, infra.

METHODS

Also provided by the subject invention are methods of using the subjecttest strips to determine the concentration of an analyte in aphysiological sample. A variety of different analytes may be detectedusing the subject test strips, where representative analytes includeglucose, cholesterol, lactate, alcohol, and the like. In many preferredembodiments, the subject methods are employed to determine the glucoseconcentration in a physiological sample. While in principle the subjectmethods may be used to determine the concentration of an analyte in avariety of different physiological samples, such as urine, tears,saliva, and the like, they are particularly suited for use indetermining the concentration of an analyte in blood or blood fractions,e.g. blood derived samples, and more particularly in whole blood.

In practicing the subject methods, the first step is to apply a quantityof the physiological sample to the test strip, where the test strip isdescribed supra. The amount of physiological sample, e.g. blood, that isapplied to the test strip may vary, but generally ranges from about 2 μLto 40 μL, usually from about 5 μL to 20 μL. Because of the nature of thesubject test strip, where blood glucose concentration if of interest,the blood sample size that is applied to the test strip may berelatively small, ranging in size from about 2 μL to 40 μL, usually fromabout 5 μL to 20 μL. Where blood is the physiological sample, bloodsamples of a variety of different hematocrits may be assayed with thesubject methods, where the hematocrit may range from about 20% to 65%,usually from about 25% to 60%.

Following application of the sample to the test strip, the sample isallowed to react with the members of the signal producing system toproduce a detectable product that is present in an amount proportionalto the initial amount present in the sample. The amount of detectableproduct, i.e. signal produced by the signal producing system, is thendetermined and related to the amount of analyte in the initial sample.In certain embodiments, automated instruments that perform the abovementioned detection and relation steps are employed. The above describedreaction, detection and relating steps, as well as instruments forperforming the same, are further described in U.S. Pat. Nos. 4,734,360;4,900,666; 4,935,346; 5,059,394; 5,304,468; 5,306,623; 5,418,142;5,426,032; 5,515,170; 5,526,120; 5,563,042; 5,620,863; 5,753,429;5,573,452; 5,780,304; 5,789,255; 5,843,691; 5,846,486; 5,968,836 and5,972,294; the disclosures of which are herein incorporated byreference. In the relation step, the derived analyte concentration takesinto account the constant contribution of competing reactions to theobserved signal, e.g. by calibrating the instrument accordingly.

Because of the presence of hemolyzing agent on the test strips employedin the subject methods, the results that are obtained by the subjectmethods are substantially, if not completely, free of analytical errorthat arises in configurations that lack a hemolyzing agent on the teststrip, where the analytical error is a result of the presence oferythrocyte based interfering components, e.g. catalase, hemoglobin,glutathione peroxidase and the like. As such, the subject methods aresubstantially, if not completely, free of the hematocrit effect whichcan introduce analytical error to analyte measurements made with otherdetection devices and protocols. In addition, because of the presence ofthe hemolyzing agent(s) on the test strip, results are obtained in arapid manner, where results can be obtained in less than about 20seconds, usually less than about 30 seconds and more usually less thanabout 40 seconds following application of the sample to the test strip.

KITS

Also provided by the subject invention are kits for use in practicingthe subject methods. The kits of the subject invention at least includea reagent test strip that includes a hemolyzing agent, as describedabove. The subject kits may further include a means for obtaining aphysiological sample. For example, where the physiological sample isblood, the subject kits may further include a means for obtaining ablood sample, such as a lance for sticking a finger, a lance actuationmeans, and the like. In addition, the subject kits may include a controlsolution or standard, e.g. a glucose control solution that contains astandardized concentration of glucose. In certain embodiments, the kitsalso include an automated instrument, as described above, for detectingthe amount of product produced on the strip following sample applicationand related the detected product to the amount of analyte in the sample.Finally, the kits include instructions for using the subject reagenttest strips in the determination of an analyte concentration in aphysiological sample. These instructions may be present on one or moreof the packaging, a label insert, containers present in the kits, andthe like.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

A. Preparation of Test Strips

The porous side of a 0.35 μm polysulfone membrane (reactionmatrix—obtained from U.S. Filter, San Diego, Calif.) was submerged inthe aqueous dip shown in Table 1 until saturated. It was removed fromthe dip and the excess reagent was squeezed off with a glass rod. Thestrip was then hung inside an air circulating oven at 56° C. for about10 minutes to dry, after which time the strip was removed and dippedinto the organic dip described in Table 2 until saturated. It was thendried again as in the previous step. The resulting strip was fashionedinto the desired shape for testing.

TABLE 1 Ingredient Amount H₂O 25 mL Citric Acid 282 mg Trisodium Citrate348 mg Mannitol 250 mg EDTA 21 mg Gantrez (obtained from GAF, New York,New York) 112.5 mg Crotein (obtained from CRODA, New York, New York) 360mg Glucose Oxidase (126 U/mg) 234.5 mg Horse Radish Peroxidase (505U/mg) 62 mg Carbapol 910 (0.11 mg/mL in acetonitrile) (obtained 1.25 mLfrom BFGoodrich, Clevelend Ohio) 0.1 M disodium citrate 3.75 mL

TABLE 2 Ingredient Amount MeOH/EtOH/H₂O (17.5/52.5/30) 9.54 mL MBTHSB38.8 mg Meta[3-methyl-2-benzothiazolinone hydrazone]N- sulfonylbenzenesulfonate monosodium ANS 54 mg MAPHOS 60A (20% in the abovesolvent) 0.46 mL (PPG/Mazer, Gurnee, Illinios) ⊥hemolyzing surfactant orcontrol 0 to 50 mg ⊥Hemolyzing Surfactants: control = 0 g = 0%N-hexadecyltrimethylammonium chloride(CTAC) = 7.5 mg = 0.075%N-hexadecyltrimethylammonium chloride(CTAC) = _(—)25 mg = 0.25% TritonX-100 25 mg = 0.25% Brij 58 = 50 mg = 0.5% Lubrol PX = 50 mg = 0.5%B. Testing

SureStep® strip configurations were used for testing of glucoseresponse. Reflectance data was collected on modified SureStep® meters.Reflectance spectral data was acquired using Macbeth Color Eye(GretagMacbeth, New Windsor, N.Y.). Blood samples are as noted.

III. Results

FIG. 1 shows the effect of hemolysis on the meter response. FIG. 1demonstrates that most of the decrease in color formation due tocompeting reactions is produced by hemolysis in the rang of 0 to 8% andthe response remains constant at the range of 10 to 20% hemolysis. Byadding a certain amount of hemolyzing surfactant to the reagentformulation, one can ensure that blood samples applied to the strip arehemolyzed in the range of 10 to 20% across the range of potentialhematocrit. This range of hemolysis allows for analyte calibration thatis unaffected by the level of hematocrit. See FIGS. 2 a and 2 b.Endpoint is achieved faster in the presence of hemolysate, since some ofthe hydrogen peroxide (produced by the peroxidase reaction), is beingconsumed by reactions with hemolysate components. See FIGS. 3 and 4.FIGS. 3 and 4 provide the observed test response and reaction kineticsfor a control and 0.25% CTAC strip at a whole blood concentration of 390mg/dL. FIGS. 5 and 6 provide the observed test response and reactionkinetics for a control and 0.25% Triton X-100 strip at a whole bloodconcentration of 390 mg/dL. FIGS. 7 and 8 provide the observed testresponse and reaction kinetics for a control and 0.50% Brij-58 strip ata whole blood concentration of 390 mg/dL; while FIG. 9 provides theobserved test response and reaction kinetics for a 0.50% Lubrol PX stripat a whole blood glucose concentration of 390 mg/dL. FIGS. 10 and 11demonstrate the hemolyzing effect of the CTAC surfactants as indicatedby a higher absorbance at the hemoglobin's Soret band (around 400 nm) inthe presence of CTAC. Visual confirmation of the test results is abeneficial feature offered by the SureStep system. FIG. 10 shows thathemolysis at the range required in this invention does not causeincreased blood color (red appearance) in the visual range even whenhigh hematocrit sample is applied to the strip, and therefore will notinterfere with the visual confirmation of the test results.

It is evident from the above results and discussion that the subjectinvention provides for a significant improvement in hematocritperformance with respect to analytical error results from erythrocytebased interfering components. In addition, the subject inventionprovides for these improved results without requiring an initially largephysiological sample or a long assay time. As such, the subjectinvention represents a significant contribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A kit for use in determining a concentration of an analyte in aphysiological sample, said kit comprising: (a) a reagent test stripcomprising: (i) a porous matrix; (ii) at least one member of an analyteoxidation signal producing system; and (iii) at least one hemolyzingagent in an amount sufficient to produce 5–20% by volume hemolysate inthe sample; and (b) at least one of: (i) a means for obtaining saidphysiological sample; and (ii) an analyte standard.
 2. The kit accordingto claim 1, wherein said analyte is glucose.
 3. The kit according toclaim 1, wherein said physiological sample is blood.
 4. The kitaccording to claim 1, wherein said means for obtaining saidphysiological sample is a lance.
 5. The kit according to claim 1,wherein said kit further comprises an automated instrument for detectinga signal produced by said signal producing system and relating saiddetected signal to the concentration of analyte in the sample.
 6. Thekit according to claim 1, wherein said at least one member of an analyteoxidation signal producing system comprises an enzyme that oxidizes ananalyte to produce hydrogen peroxide.
 7. The kit according to claim 6,wherein said signal producing system further comprises an enzyme thatconverts at least one substrate into a chromogenic product in thepresence of hydrogen peroxide.
 8. The kit according to claim 1, whereinsaid hemolyzing agent is present in an amount sufficient to producehemolysis which is equivalent to hemolysate concentration in aphysiological sample applied to said porous matrix in an amount rangingfrom about 10 to 20% by volume.